Abstract
SARS-CoV-2 is the causative agent behind the COVID-19 pandemic, responsible for over 170 million infections, and over 3.7 million deaths worldwide. Efforts to test, treat and vaccinate against this pathogen all benefit from an improved understanding of the basic biology of SARS-CoV-2. Both viral and cellular proteases play a crucial role in SARS-CoV-2 replication. Here, we study proteolytic cleavage of viral and cellular proteins in two cell line models of SARS-CoV-2 replication using mass spectrometry to identify protein neo-N-termini generated through protease activity. We identify previously unknown cleavage sites in multiple viral proteins, including major antigens S and N: the main targets for vaccine and antibody testing efforts. We discover significant increases in cellular cleavage events consistent with cleavage by SARS-CoV-2 main protease, and identify 14 potential high-confidence substrates of the main and papain-like proteases. We show that siRNA depletion of these cellular proteins inhibits SARS-CoV-2 replication, and that drugs targeting two of these proteins: the tyrosine kinase SRC and Ser/Thr kinase MYLK, show a dose-dependent reduction in SARS-CoV-2 titres. Overall, our study provides a powerful resource to understand proteolysis in the context of viral infection, and to inform the development of targeted strategies to inhibit SARS-CoV-2 and treat COVID-19.
Highlights
SARS-CoV-2 is the causative agent behind the COVID-19 pandemic, responsible for over 170 million infections, and over 3.7 million deaths worldwide
Viral proteins can be the targets of cellular proteases, with the most prominent example for coronaviruses being the cleavage of the spike glycoprotein by the cellular proteases FURIN, TMPRSS2, and Cathepsins[10,11,15,16], but the exact cleavage sites within spike for most of these individual cellular proteases are not yet characterised
Vero E6 cells are an African Green Monkey kidney cell line commonly used for the study of a range of viruses, including SARS-CoV-2 which replicates in this cell line to high titres
Summary
SARS-CoV-2 is the causative agent behind the COVID-19 pandemic, responsible for over 170 million infections, and over 3.7 million deaths worldwide. Treat and vaccinate against this pathogen all benefit from an improved understanding of the basic biology of SARS-CoV-2 Both viral and cellular proteases play a crucial role in SARS-CoV-2 replication. In-depth characterisation of the virus and its interactions with the host cell can inform current and next-generation efforts to test, treat, and vaccinate against SARS-CoV-2 Past efforts in this area have included the proteome, phosphoproteome, ubiquitome, and interactome of SARS-CoV-2 viral proteins and infected cells[3,4,5,6,7,8,9]. Proteolytic cleavage plays a crucial role in the life cycle of SARS-CoV-2, and most positive-sense RNA viruses Inhibitors targeting both viral and cellular proteases have previously shown the ability to inhibit SARS-CoV-2 replication in cell culture models[10,11,12,13]. Mass spectrometry has particular advantages for investigation of proteolytic cleavage as analysis can be conducted in an unbiased manner, and identify the substrate, and the precise site of proteolytic cleavage[22]
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